Cysts derive from renal tubules and may progressively expand to several centimeters in diameter; in a sense they are benign neoplastic tumors. The long-term objective of this application is to determine the aberrant mechanisms responsible for directing epithelial cells of normal and genetically abnormal kidney tubules to become cysts rather than terminally differentiated renal tubules. The applicants will test the general hypothesis that renal cysts of acquired and hereditary etiologies share a developmental abnormality in common, that of being locked in an altered state of differentiation and perpetual radial proliferation in which the cells are morphologically and functionally less mature than those of terminally differentiated tubular epithelium; the immature cyst phenotype is unable to respond to exogenous differentiating agents and fails to down-regulate cyclic-AMP dependent transepithelial chloride secretion through the cystic fibrosis transmembrane conductance regulator (CFTR). There are six Specific Aims: 1) use an in vitro culture method to observe the differentiation of cysts grown in collagen matrix into tubule-like structures, and determine the effect and mechanism of action of fibroblast-conditioned medium and fibroblast co-culture on embedded cells obtained from the walls of established cysts in hereditary and acquired human cystic disorders, in comparison to normal tubule epithelial cells; 2) determine the effect of fibroblast-conditioned medium and fibroblast co-culture on renal epithelial cells cultured from mice and rats with heritable forms of recessive (C57BL/6J-cpk, DBA-pcy) and dominant (HAN:SPRD-cy) renal disease; 3) identify by subtraction cloning the genes uniquely expressed in response to fibroblast-dependent differentiation of MDCK cysts into a tubular phenotype; examine the expression of these genes in spontaneous renal cystic disorders and in in vitro models of cysts derived from human renal cells; 4) determine with polarized cultures of human autosomal dominant PKD, autosomal recessive PKD, acquired cystic kidney disease and human kidney cortex cells, and polarized primary cultures of C57BL/6J-cpk, DBA-pcy, and HAN:SPRD-cy kidney cells the magnitude of chloride secretion in response to secretogogues, and correlate these with contemporaneous changes in the level of intracellular cyclic AMP; 5) determine with in situ and Northern hybridization and immunohistochemistry the extent of CFTR expression: a) in human adult intact kidneys and cells cultured from ADPKD, ARPKD, ACKD, HKC; and b) in fetal, neonatal and adult intact kidneys and cultured cells from C57BL/6J- cpk, DBA-pcy, HAN:SPRD-cy and normal animals of the same strains; and 6) determine in cultured cells grown as cysts in collagen matrix the extent to which CFTR and SGP-2 expression is downregulated in response to soluble fibroblast differentiating factors. Successful completion of this research will determine the extent to which extrinsic signalling mechanisms direct normal human kidney cells to form renal tubules, and will identify specific cellular elements that are developmentally misregulated in aberrant tubulogenesis that leads to cyst formation and expansion.